MEMBRANE-BOUND D-GLUCONATE DEHYDROGENASE FROM PSEUDOMONAS-AERUGINOSA - PURIFICATION AND STRUCTURE OF CYTOCHROME-BINDING FORM

Abstract

A membrane-bound D-gluconate dehydrogenase [EC 1.1.99.3] was solubilized from membranes of P. aeruginosa and purified to a homogeneous state with the aid of detergents. The solubilized enzyme was a monomer in the presence of at least 0.1% Triton X-100, having a MW of 138,000 on polyacrylamide gel electrophoresis or 124,000-131,000 on sucrose density gradient centrifugation. In the absence of Triton X-100, the enzyme became dimeric, having a MW of 240,000-260,000 on sucrose density gradient centifugation. Removal of Triton X-100 caused a decrease in enzyme activity. Enzyme activity was stimulated by addition of phospholipid, particularly cardiolipin, in the presence of Triton X-100. The enzyme had a cytochrome c1, c-554(551), which might be a diheme cytochrome, and it also contained a covalently bound flavin but not ubiquinone. In the presence of sodium dodecyl sulfate, the enzyme was dissociated into 3 components with MW of 66,000, 50,000 and 22,000. The components of 66,000 and 50,000 daltons corresponded to a flavoprotein and cytochrome c1, respectively, but that of 22,000 dalton remained unclear as to its function.